1. Field of the Invention
The present invention provides methods and reagents for determining an individual's genotype at the .sup.G .gamma.-globin locus with respect to a set of three alleles using sequence-specific oligonucleotide probes. The invention enables one to type samples from a variety of sources, including samples comprising RNA or cDNA templates, and can be applied to amplified nucleic acids containing the target polymorphic region. The three-allele system provides substantially more information than the prior art bi-allelic system based on the polymorphic HindIII site. The present typing system facilitates typing tissue for determining individual identity and is especially useful in the field of forensic science.
2. Description of Related Art
The .beta.-related globin proteins are encoded by genes on the short arm of human chromosome 11. The .sup.G .gamma.-globin and .sup.A .gamma.globin genes respectively code for the .sup.G .gamma.- and .sup.A .gamma.-polypeptide chains of .alpha..sub.2.sup.G .gamma..sub.2 and .alpha..sub.2.sup.A .gamma..sub.2, the major species of fetal hemoglobin produced by the fetal liver. The .beta.- and .delta.-globin genes are activated around birth, while the two .gamma.-globin genes are inactivated. The two adult hemoglobins, produced in the bone marrow, are .alpha..sub.2 .beta..sub.2 and .alpha..sub.2 .delta..sub.2. The nucleotide sequences of the .sup.G .gamma.-globin and .sup.A .gamma.-globin genes were first described by Slightom et al., 1980, Cell 21:627-638. Each gene consists of three exons and two intervening sequences (IVS).
The .sup.G .gamma.-globin and .sup.A .gamma.-globin genes have been classified by variant patterns of restriction endonuclease cleavage site number and location. In particular, a HindIII site polymorphism has been discovered (Jeffreys, 1979, Cell 18:1-10) near the 3' end of the second IVS.
Nucleotide sequence variability corresponding to the HindIII site polymorphism has been observed in the study of gene conversion in the two fetal globin genes (Shiokawa et al., 1989, J. Biochem. 105:184-189, incorporated herein by reference). Probes for the sequences corresponding to the HindIII polymorphism have been used in a study of linkage between the .sup.G .gamma.-globin and parathyroid loci (Cui et al., 1989, Proc. Natl. Acad. Sci. 6:9389-9393). In both of these studies, the observed HindIII site polymorphism corresponds to the present A and B alleles.
Identification of individuals is possible with genetic typing. The use of polymerase chain reaction (PCR) amplification and nucleotide probes to detect even single nucleotide changes in a gene sequence has revolutionized the field of forensic serology (see Reynolds and Sensabaugh 1991, Anal. Chem. 63:2-15). With PCR and other nucleic acid amplification methods, DNA typing can now be done with samples that contain insufficient DNA for typing by any other means; single hairs, for example, provide enough DNA for PCR-based DNA typing. (Higuchi et al., 1988, Nature 332:543-546).